Secretion of a protein from a cell involves the recognition of the protein as one destined for export and followed by its passage through a membrane to the outside. These basic steps take place while the proteins are nascent and at the plasma membrane (PM) in procaryotes, the endoplasmic reticulum (Er) in eucaryotes. It is not known how a polypeptide chain traverses a membrane, although it undoubtedly necessitates a conformational change which may be brought about by various agents. Since eucaryotes secrete glycoproteins, it is thought that the covalently bound carbohydrate plays a role in the process. The goal of the research proposed herein is to determine the way in which secretory proteins are recognized and are enabled to cross a membrane in the fungus Aspergillus niger. This organism is easily cultured under a variety of conditions and it produces and secretes a number of well characterized glycoprotein enzymes. The membrane-bound form of one of these, an alpha-glucosidase (alpha-gluE), which may be a secretion intermediate, will be purified and analyzed to determine its point of attachment and orientation in the PM. Its insertion into the PM will be studied by isolating membrane-bound polysomes (MBP) and placing them into a cell-free protein synthesizing system followed by detection of inserted alpha-gluE by electrophoresis in polyacrylamide gels under denaturing conditions. Also, MBP will be reconstituted by mixing purified mRNA, ribosomes, and membrane from A. niger with other heterologous components necessary for cell-free translation to discover the mode and kinetics of an inhibitor of glycosylation, will allow a determination of the importance of carbohydrate addition to membrane insertion.